INTRODUCTION: We have shown that mast cell degranulation is dependent on a rise in intracellular calcium (calcium signal)in conjunction with activation of protein kinase (PK) C and phospholipase(PL)D. We have focussed on PLD in recent years because least is known about this enzyme. PLD is activated in mast cells by antigen and other stimulants and is the major source of biologically lipids such as phosphatidic acid and diglycerides. PLD appears to play an essential role in degranulation because PLD activation is closely correlated with degranulation under a variety of experimental conditions which include pharmacological disruption (Chadi et al., Mol. Immunol. 38:1269, 2001)and knock down of PLD with iRNAs (Peng and Beaven, J. Immunol.174:5201,2005). Furthermore, the two known isoforms of PLD, PLD1 and PLD2, reside in different locations within the cell. PLD1 is associated with Golgi and granule membranes and PLD2 with the plasma membrane (Choi et al., J. Immunol. 278:12039,2002). Both isoforms regulate different phases of degranulation in mast cells, granule-associated PLD1 in the translocation of granules to the plasma membrane and plasma membrane-associated PLD2 in the fusion of granules with the plasma membrane. However, the mechanisms by which PLD is activated and the downstream PLD-dependent signaling events remain largely undefined. These include possible links to entry of Ca2+ via phosphatidylinositol 5-kinase (PI5K) and sphingosine kinase (SK). [unreadable] [unreadable] OBJECTIVES: We are pusuing two lines of investigation in regard to PLD: the first is to elucidate the mechanisms of activation of PLD and the second is to identify downstream events that are regulated by PLD. PLD2 is of particular interest because its location at the plasma membrane makes this isoform accessible to FcepsilonRI-associated Src kinases. As reported last year, we found that PLD2, but not PLD1, is phosphorylated by the Src kinases, Fyn and Fgr, and that this phosphorylation regulates PLD2 activation and degranulation (Choi et al., Mol. Cell. Biol. 24:6980,2004). These earlier studies also revealed that PLD2 co-localized with "lipid rafts" and that phosphorylation of PLD2 was dependent on integrity of these "lipid rafts" (unpublished data). In the current period we have investigated whether PLD itself is required for the functional integrity of "lipid rafts" and the activation of downstream kinases such as Syk which plays a critical role in initiating several signaling cascades in mast cells. Also, following our observation that diglyceride-dependent isoforms of PKC are highly regulated by PLD (Peng and Beaven, J. Immunol. 174:5201,2005), we are now investigating the role of PLD in the activation of PI5K and SK because PLD products are potential activators of these enzymes. As both PI5K and SK have been implicated in the generation of a calcium signal, this topic has become an important component of our recent research.[unreadable] [unreadable] PLD AND LIPID RAFT FUNCTION: Suppression of PLD function, either with primary alcohols or siRNAs, results in dispersal of lipid raft components including LAT, Thy1, and GPI and suppression of the translocation of the IgE receptor (FceRI) and its associated tyrosine kinases, Lyn and Syk, into lipid rafts following antigen stimulation. Downstream phosphorylation events are also blocked. Consistent results were obtained whether cells were examined by use of fluorescent tagged molecules and confocal microscopy or by classical membrane fractionation techniques. These techniques indicate that tagged PLD2 also colocalizes with lipid raft constituents, a process that is prevented by the lipid raft dispersing agent, methyl beta-cyclodextrin. These observations suggest that not only is PLD2 activation dependent on lipid raft integrity but that this activation contributes to the functional organization of lipid rafts (unpublished data).[unreadable] [unreadable] PLD2 INTERACTS WITH SYK AND REGULATES SYK ACTIVITY AND DOWNSTREAM EVENTS: PLD2 associates with and promotes activation of Syk which, as noted above is a key enzyme in mast cell activation. Antigen stimulation results in co-localization of Syk with PLD2 within membrane lipid rafts as indicated by co-immunoprecipitation and confocal microscopy. This association is dependent on the initial phosphorylation of Syk by Lyn and binding of PLD2 to Syk via its Phox homology (PX) domain to induce further phosphorylation and activation of Syk and its downstream targets, LAT and SLP76. Paradoxically, expression studies indicated that a catalytically inactive mutant of PLD2 was equally active in this regard. Overexpression of PLD2, or the PLD2 mutant, enhanced antigen-induced phosphorylations of Syk and downnstream targets while expression of a PLD2 siRNA blocked these phosphorylations. Similar genetic manipulations suggested that the interaction of PLD2 with Syk was necessary for degranulation. These findings indicate a dual role for PLD2 as as a signaling molecule. It can act in a catalytic manner to produce biologically active lipids such as phosphatidic acid and diglycerides and as an adaptor molecule to facilitate signal transduction via Syk (Ref. 1).[unreadable] [unreadable] REGULATION OF PHOSPHATIDYLINOSITOL 5-KINASE, SPHINGOSINE KINASE, CALCIUM MOBILIZATION, AND DEGRANULATION BY PLD: As reported last year, the production of phosphatidic acid by PLD is the primary source of diacylglycerides in mast cells and is essential for sustained activation of diglyceride-dependent isoforms of PKC and degranulation. However, additional PLD-dependent processes appear to be necessary for antigen-mediated degranulation. One such process could be the activation of Syk as described above(Peng and Beaven, J. Immunol. 174:5201,2005).[unreadable] [unreadable] Pharmacological and genetic studies now show that PLD is linked to the activation of PI5K and SK in addition to PKC. PI5K catalyzes the conversion of phosphatidylinositol 4-phosphate (PIP) to phosphatidylinositol 4,5-bisphosphate (PIP2). SK kinase phosphorylates sphingosine to form sphingosine 1-phosphate (S1P) (ref. 2). Reports indicate that PIP2 regulates translocation of the TRPC5 Ca2+ channel protein from proximal vesicles to the plasma membrane a. S1P acting in conjunction with IP3 promotes release of Ca2+ from intracellular stores and subsequently influx of external Ca2+. In our hands, knockdown of PLD2 or PI5K blocks entry of Ca2+, as do SK inhibitors, with minimal effect on release of Ca2+ from intracellular stores in stimulated mast cells. These studies clearly demonstrate that PI5K and SK regulate Ca2+ influx rather than Ca2+ release. We are currently defining the mechanisms inolved in studies with inhibitory RNAs and genetically deficient mast cells (see ref.3). [unreadable] [unreadable] IDENTIFICATION OF CALCIUM CHANNELS AND REGULATORY PROTEINS FOR CALCIUM ENTRY[unreadable] Degranulation of mast cells is dependent on emptying of intracellular stores of Ca2+ and the ensuing influx of external Ca2+, also referred to as store-operated calcium entry (SOCE). The well characterized calcium release-activated calcium current (CRAC) has been identified as a primary candidate for mediating SOCE. We were sceptical that CRAC was the sole mechanism for the entry of Ca2+ because Sr2+ and other divalent cations also permeate and support degranulation in stimulated mast cells (see previous reports in this series). We find that overexpression of STIM1 and Orai1, which were recently shown to be essential components of CRAC, allows entry of Ca2+ but not Sr2+ in stimulated mast cells. However, influx of Ca2+ and Sr2+ as well as degranulation are dependent on the presence of TRPC5 (see previous section), in addition to STIM1 and Orai1, as demonstrated by siRNA knock down of each of these proteins. Our observations suggest that TRPC5 associates with STIM1 and Orai1 in a stoichiometric manner to enhance entry of Ca2+ or Sr+ to generate a signal for degranulation (manuscript submitted).